Antibacterial Polyketides from the Deep-Sea Cold-Seep-Derived Fungus Talaromyces sp. CS-258

Thirty-two fungal polyketide derivatives, including eleven new compounds, namely (3R,5′R)-5-hydroxytalaroflavone (1), talaroisochromenols A–C (3, 5, and 11), (8R,9R,10aR)-5-hydroxyaltenuene (13), (8R,9R,10aS)-5-hydroxyaltenuene (14), (8R,9S,10aR)-5-hydroxyaltenuene (15), nemanecins D and E (25 and 26), 2,5-dimethyl-8-iodochromone (27), and talarofurolactone A (29), together with one new naturally occurring but previously synthesized metabolite, 6-hydroxy-4-methoxycoumarin (28), were isolated and identified from the deep-sea cold-seep-derived fungus Talaromyces sp. CS-258. Among them, racemic ((±)-11) or epimeric (13–15, 25, and 26) mixtures were successfully separated by chiral or gradient elution HPLC. Meanwhile, compound 27 represents a rarely reported naturally occurring iodinated compound. Their planar structures as well as absolute configurations were determined by extensive analysis via NMR, MS, single-crystal X-ray diffraction, Mosher’s method, and ECD or NMR calculation (with DP4+ probability analysis). Possible biosynthetic routes of some isolated compounds, which are related to chromone or isochromone biosynthetic pathways, were put forward. The biological analysis results revealed that compounds 7, 9, 10, 18–22, 24, 30, and 31 showed broad-spectrum antibacterial activities against several human and aquatic pathogens with MIC ranges of 0.5–64 μg/mL.


Introduction
Polyketides are a big family of secondary metabolites generally produced from a common origin of acetyl-CoA polymerization by connecting acetic acid units via condensation reactions and intermediated by a non-reducing group of iterative polyketide synthases (PKSs) [1,2].Fungal polyketides have attracted considerable attention due to their unique molecular scaffolds and diverse pharmacological activities [3][4][5].Among them, a series of fungal polyketides, such as griseofulvin, brefeldin, and hypomycetin, have been selected as promising candidates for new antibiotic lead compounds and possess great commercial values [1,3,5].
In the course of forwarding our program on discovering bioactive metabolites from deep-sea cold seep-derived fungi [6,15,16], a fungal strain Talaromyces sp.CS-258, which was isolated from a mussel sample collected in a cold seep area in the northeast of the South China Sea at a depth of −1200 m, displayed diverse classes of polyketide derivatives and exhibited antimicrobial activity in a preliminary screening.Intensive chemical investigations on the fermentation broth of the fungus resulted in the isolation and identification of 32 polyketides (Figure 1), including 11 new chromone-or isochromone-derived analogs (compounds 1, 3, 5, 11, 13-15, 25-27, and 29) and 1 new naturally occurring coumarin (28) that was previously obtained by chemical synthesis [18].The isolated compounds were assayed for their antimicrobial activities against pathogenic microbes, which demonstrated that eleven polyketides, including 7, 9, 10, 18-22, 24, 30, and 31, possessed broad-spectrum antibiotic activities against human and aquatic pathogenic bacteria with MIC values ranging from 0.5 to 64 μg/mL.Herein, the details of isolation, structural elucidation, plausible biosynthetic pathways, and bioactivity assays of these compounds are described.The structure-activity relationships (SARs) for these antibacterial polyketides have been briefly discussed in this paper.

Structure Elucidation
The molecular formula of compound 1 was given as C 13 H 10 O 6 from HRESIMS data with nine degrees of unsaturation.A comprehensive analysis of its 1 H, 13 C, and DEPT NMR spectra (Table 1) revealed great similarity to talaroflavone (2), a previously described compound isolated from the sponge-derived fungus Alternaria sp.F49 [19].However, the signals of a methoxy group at δ C/H 55.8/3.75 at C-5 in the NMR spectra of 2 were absent in those of 1.In comparison to 2, obvious upfield shifts for C-5 in 1 were detected.These differences suggested that compound 1 was a 5-demethylation derivative of 2, which was further supported by the HMBC correlations of H-4/H-6 to C-5 (Figure 2).From a biosynthetic point of view, the stereoscopic configuration of 1 was assumed to be the same as that of 2. After slow evaporation of the MeOH solvent, single crystals of 2 were obtained.The relative configuration of 2 was thus assigned by the result of a single-crystal X-ray diffraction experiment using Cu Kα radiation as 3S*, 5 ′ S* (Figure 3).Accordingly, the relative configuration of 1 was also established as 3S*, 5 ′ S*, the same as 2. The absolute configuration of 1 was further studied by the time-dependent density functional (TDDFT) ECD calculation.The calculated ECD curve of (3R,5 ′ R)-1 agreed well with the experimental ECD curve of 1 (Figure 4), which ascertained compound 1 as (3R,5 ′ R)-5-hydroxytalaroflavone.
No. The protonated molecular ion peak at m/z 261.0398 [M − H] -in HRESIMS revealed the molecular formula of C 13 H 10 O 6 for 3 with nine degrees of unsaturation.As shown in Table 1, the 1 H NMR spectrum exhibited signals for two singlet aromatic protons (δ H 3.26 and 2.94), one doublet methylene (δ H 3.12 and 2.88), and one singlet methyl (δ H 1.70).The 13 C and DEPT NMR spectra data of 3 indicated the presence of 13 carbon signals, which were sorted into one methyl, one methylene, two methines, and nine quaternary carbons.Detailed analysis of the NMR data revealed that the structure of 3 was similar to that of compound 4, an altenusin analog bearing a 6/6/5 tricyclic ring skeleton obtained from the mangrove endophytic fungus Alternaria sp.SK6YW3L [20].However, resonances for a methoxy group of 4 were absent in the NMR spectra of 3, which suggested that 3 was a 5-demethylated derivative of 4. The above deduction was supported by HMBC correlation from H-4 to C-5 (Figure 2).The absolute configuration of 3 was established as 9aS based on the quantum chemical calculation of ECD (Figure 4) and nominated as talaroisochromenol A.
Compound 5 was afforded as a yellow oil, and the molecular formula of C 13 H 12 O 6 for 5 was assigned by its positive HRESIMS.The NMR spectra of 5 were identical to those of 6, a known altenusin derivative with a 6/6/5 tricyclic ring [20], except for the obvious differences in the chemical shifts of CH-8, CH-9, and CH 3 -10.The above observation suggested that 5 was a new epimer of 6 (epimeric at the C-9 position), which was further proved by the NOESY correlations from H-7 to H-9 and from H 3 -10 to H-8 (Figure 5).Hence, the absolute configuration of 5 was assigned as 7R, 8S, 9R, which was further confirmed by comparison of its calculated ECD for 7R, 8S, 9R-5 with the measured ECD spectra (Figure 4).Thus, compound 5 was acknowledged as a new compound, namely talaroisochromenol B.
Compound 11 possessed a molecular formula of C 12 H 10 O 6 as established from its HRESIMS spectrum, accounting for eight degrees of unsaturation.In the 1 H NMR spectra, resonances for two phenolic hydroxyl groups (δ H 11.08 and 10.99), two meta-substituted aromatic protons (δ H 6.62, d, J = 2.2 Hz; δ H 6.46, d, J = 2.2 Hz), one singlet oxymethine (δ H 5.49), a chemically nonequivalent methylene (δ H 3.26 and 2.94), and one singlet methyl (δ H 1.51) were observed.The investigation of 13 C NMR and HSQC spectral data displayed attribution signals of two lactone carbonyl groups, six aromatic carbons (including two methines and four quaternary carbons), one oxygenated quaternary carbon, one oxygenated methine, one methylene, and one methyl.These data demonstrated great similarity to those of 12, a known polyketide yielded from an endolichenic fungus Ulocladium sp.[21], except for the absence of a methoxy group at C-8 (Table 2).Compared to 12, the obvious upfield shift of C-8 in 11 was observed.Thus, 11 was determined as an 8-hydroxylated derivative of 12.

Structure Elucidation
The molecular formula of compound 1 was given as C13H10O6 from HRESIMS data with nine degrees of unsaturation.A comprehensive analysis of its 1 H, 13 C, and DEPT NMR spectra (Table 1) revealed great similarity to talaroflavone (2), a previously described compound isolated from the sponge-derived fungus Alternaria sp.F49 [19].However, the signals of a methoxy group at δC/H 55.8/3.75 at C-5 in the NMR spectra of 2 were absent in those of 1.In comparison to 2, obvious upfield shifts for C-5 in 1 were detected.These differences suggested that compound 1 was a 5-demethylation derivative of 2, which was further supported by the HMBC correlations of H-4/H-6 to C-5 (Figure 2).From a biosynthetic point of view, the stereoscopic configuration of 1 was assumed to be the same as that of 2. After slow evaporation of the MeOH solvent, single crystals of 2 were obtained.The relative configuration of 2 was thus assigned by the result of a single-crystal X-ray diffraction experiment using Cu Kα radiation as 3S*, 5'S* (Figure 3).Accordingly, the relative configuration of 1 was also established as 3S*, 5'S*, the same as 2. The absolute configuration of 1 was further studied by the time-dependent density functional (TDDFT) ECD calculation.The calculated ECD curve of (3R,5'R)-1 agreed well with the experimental ECD curve of 1 (Figure 4), which ascertained compound 1 as (3R,5'R)-5-hydroxytalaroflavone.

Structure Elucidation
The molecular formula of compound 1 was given as C13H10O6 from HRESIMS with nine degrees of unsaturation.A comprehensive analysis of its 1 H, 13 C, and D NMR spectra (Table 1) revealed great similarity to talaroflavone (2), a previousl scribed compound isolated from the sponge-derived fungus Alternaria sp.F49 [19].ever, the signals of a methoxy group at δC/H 55.8/3.75 at C-5 in the NMR spectra of 2 absent in those of 1.In comparison to 2, obvious upfield shifts for C-5 in 1 were dete These differences suggested that compound 1 was a 5-demethylation derivative which was further supported by the HMBC correlations of H-4/H-6 to C-5 (Figure 2). a biosynthetic point of view, the stereoscopic configuration of 1 was assumed to b same as that of 2. After slow evaporation of the MeOH solvent, single crystals of 2 obtained.The relative configuration of 2 was thus assigned by the result of a single-c X-ray diffraction experiment using Cu Kα radiation as 3S*, 5'S* (Figure 3).Accord the relative configuration of 1 was also established as 3S*, 5'S*, the same as 2. The abs configuration of 1 was further studied by the time-dependent density functional (TD ECD calculation.The calculated ECD curve of (3R,5'R)-1 agreed well with the ex mental ECD curve of 1 (Figure 4), which ascertained compound 1 as (3R,5'R)-5-hyd talaroflavone.The protonated molecular ion peak at m/z 261.0398 [M − H] -in HRESIMS re the molecular formula of C13H10O6 for 3 with nine degrees of unsaturation.As sh Table 1, the 1 H NMR spectrum exhibited signals for two singlet aromatic protons and 2.94), one doublet methylene (δH 3.12 and 2.88), and one singlet methyl (δH 1.7 13 C and DEPT NMR spectra data of 3 indicated the presence of 13 carbon signals were sorted into one methyl, one methylene, two methines, and nine quaternary c Detailed analysis of the NMR data revealed that the structure of 3 was similar to compound 4, an altenusin analog bearing a 6/6/5 tricyclic ring skeleton obtained fr mangrove endophytic fungus Alternaria sp.SK6YW3L [20].However, resonance methoxy group of 4 were absent in the NMR spectra of 3, which suggested that 3 w demethylated derivative of 4. The above deduction was supported by HMBC corr from H-4 to C-5 (Figure 2).The absolute configuration of 3 was established as 9aS on the quantum chemical calculation of ECD (Figure 4) and nominated as ta chromenol A.
Compound 5 was afforded as a yellow oil, and the molecular formula of C13H 5 was assigned by its positive HRESIMS.The NMR spectra of 5 were identical to t 6, a known altenusin derivative with a 6/6/5 tricyclic ring [20], except for the obvio ferences in the chemical shifts of CH-8, CH-9, and CH3-10.The above observatio gested that 5 was a new epimer of 6 (epimeric at the C-9 position), which was proved by the NOESY correlations from H-7 to H-9 and from H3-10 to H-8 (Fig Hence, the absolute configuration of 5 was assigned as 7R, 8S, 9R, which was further firmed by comparison of its calculated ECD for 7R, 8S, 9R-5 with the measured ECD tra (Figure 4).Thus, compound 5 was acknowledged as a new compound, namely ta sochromenol B.    The relative configuration of 11 was determined by the NOESY correlation between H-9b and H 3 -10 as 3aS*, 9aS* (Figure 5).The planar structure and relative configuration of 11 were further verified by an X-ray diffraction experiment (Figure 3).However, compound 11 was acquired as a racemate with an optical rotation value near zero, and no obvious Cotton effect was observed from the ECD spectrum.The chiral HPLC separation of 11 on a Chiralcel IG column (hexane/isopropanol = 80:20, flow rate of 1 mL/min) succeeded in separating two isomers (Figure S2).Finally, as shown in Figure 4, the ECD computation allowed the assignment of absolute configurations for two isomers as (+)-11-(3aR, 9aR) and (−)-11-(3aS, 9aS), respectively.Thus, 11 was named talaroisochromenol C.
Compounds 13-15 were originally isolated as yellow oils and owned the same molecular formula of C 14 H 14 O 6 based on their positive HRESIMS data.The UV spectra of 13-15 showed similar characteristic absorptions for an isocoumarin chromophore at λ max 242, 282, and 323 nm [22].Subsequent interpretation of their NMR data (Table 3) revealed that compounds 13-15 were characterized as three new stereoisomers of a known analog 5-hydroxyaltenuene, which were isolated from an endophytic fungus Penicillium sp.FJ-1 of Ceriops tagal [22].The relative configurations of 13-15 were assigned by NOESY, X-ray single-crystal diffraction experiment, or NMR calculations with DP4+ probability analysis.As shown in Figure 5, NOESY correlations from Hα-10 to H-8 and H 3 -11 implied the α-orientation of CH 3 -11 and β-orientation of OH-8 in 13.Meanwhile, NOESY correlations from H 3 -11 to H-9/Hβ-10 and from H-8 to Hα-10 suggested a syn orientation of CH 3 -11 and H-9 and an opposite orientation of H-9 and H-8 in 14.The relative configurations of 13 and 14 were further confirmed by single-crystal X-ray diffraction experimental data as (8R*,9R*, 10aR*)-13 and (8S*,9S*,10aR*)-14 (Figure 3).Moreover, NOESY correlations of 15 from H 3 -11 to H-9 and Hα-10 established its relative configuration of 9S*, 10aR*.The experimental 1 H and 13 C NMR data of 15 were compared with the calculated NMR data of 15a and 15b (two possible isomers of 15, Figure 6) and matched well with those calculated for the isomer 15a (8R*,9S*,10aR*) with a DP4+ probability of 100% (Table S16).The absolute configurations of 13-15 were determined by comparing the experimental ECD data with the calculated ones (Figure 4), suggesting 13-15 to be (8R,9R,10aR)-, (8R,9R, 10aS)-and (8R,9S,10aR)-5-hydroxyaltenuenes, respectively.Notably, the orientation of the methyl group at C-11 significantly determined the ECD Cotton effects observed for 5-hydroxyaltenuene.This was supported by the observation that compounds 13 and 15, with 10aR absolute configurations, displayed positive Cotton effects at 236 nm and negative ones at 282 nm, in contrast to 14 with a 10aS absolute configuration.This was the first isolation of three new epimers of 5-hydroxyaltenuene from the fungus Talaromyces sp.      4) prominently indicated practically identical signals, suggesting that they shared the same planar structural scaffold.Both 1 H NMR spectral data of 25 and 26 exhibited characteristic resonances ascribed to two olefinic protons (represented for two trisubstituted double bands), three sp 3 -hybridized methines (including two oxygenated), two sp 3 -hybridized methylenes (including one chemically nonequivalent O-substituted), two methyl groups (as a singlet and a doublet), and three exchangeable protons.Analyses of their 13 C, DEPT, and HSQC spectrum confirmed the presence of a carbonyl group, two pairs of olefin carbons, two oxygenated methines, one sp 3 -hybridized methine, two methylene groups, two methyls, and one quaternary carbon.
The 1 H-1 H COSY and HMBC correlations of 25 and 26 shown in Figure 2 established the same planar structure, which was similar to nemanecins A-C, three azaphilone analogs isolated from the culture broth of the fungus Nemania sp.BCC 30850 [23], indicating that 25 and 26 are a pair of stereoisomers.However, the signal of a singlet methyl in the 1  H-11 as well as the observed HMBC correlations from H-9 to C-3, C-4, and C-10 and from H-11 to C-9 and C-10 (Figure 2).Thus, the planar structures of 25 and 26 were assigned.The relative configurations of compounds 25 and 26 of C-7, C-8, and C-8a were both assigned as 7S*,8R*, and 8aR* based on the NOESY analysis and scalar coupling constant data (Figure 5).Both of them displayed a NOESY correlation from H 3 -10 to H-8a, which implied that H 3 -10 and H-8a were co-facial and arbitrarily assigned in β-orientation.The large coupling constant between H-8a and H-8 (J = 10.1 Hz) confirmed the trans-relationship of these protons.
The modified Mosher's method using an NMR tube [24,25] was used to determine the absolute configuration of C-10 in 25 and 26, leading to the assignments of Sfor 25 and R-absolute configuration for 26 based on ∆δ values (Figure 7).ECD calculations were performed to further solve the absolution configurations of C-7, C-8, and C-8a in 25 and 26.The experimental ECD spectra of 25 and 26 both exhibited a strong positive Cotton effect at 214 nm and a negative one at 345 nm, which was in good agreement with the calculated ECD curve of (7S,8R,8aR,10S)-25 and (7S,8R,8aR,10R)-26 (Figure 5).The results of ECD calculations indicated that the configuration of C-10 made no contribution to the ECD Cotton effects.Thus, the structures of compounds 25 and 26 were finally identified and named nemanecins D and E, respectively.An analysis of the HRESIMS spectrum for m/z 316.9668 [M + H] + (calcd for 316.9669) determined the molecular formula of compound 27 (yellow oil) as C11H10O3I requiring seven degrees of unsaturation.Its 1 H and 13 CNMR data (Table 5) were in good accord with the NMR spectral information of the known chromone derivative 2,5-dimethychromone [26], except that the signal of the methine of C-8 resonating at δC 100.4 in the NMR spectra of 2,5-dimethychromone was replaced by a deprotonated carbon resonating at δC 75.3 in those of 27.The chemical shift of C-8 at δC 75.3 suggested the substitution by an iodine atom according to the previously reported compounds 2-iodo-5-methoxyphenol (δC 74.4)An analysis of the HRESIMS spectrum for m/z 316.9668 [M + H] + (calcd for 316.9669) determined the molecular formula of compound 27 (yellow oil) as C 11 H 10 O 3 I requiring seven degrees of unsaturation.Its 1 H and 13 CNMR data (Table 5) were in good accord with the NMR spectral information of the known chromone derivative 2,5-dimethychromone [26], except that the signal of the methine of C-8 resonating at δ C 100.4 in the NMR spectra of 2,5-dimethychromone was replaced by a deprotonated carbon resonating at δ C 75.3 in those of 27.The chemical shift of C-8 at δ C 75.3 suggested the substitution by an iodine atom according to the previously reported compounds 2-iodo-5-methoxyphenol (δ C 74.4) [27] and (aS)-6-iodofonsecinone A (δ C 79.3) [28].Thus, the structural assignment of 27 was assigned as 2,5-dimethy-8-iodochromone as shown in Figure 1, which was further confirmed by the HMBC correlations from H 3 -9 to C-2/C-3, H-3 to C-2/C-4a, and H-10 to C-4a/C-5/C-6 (Figure 2).Compound 28 was obtained as a yellow oil, and its 1 H and 13 C NMR spectra data (Table 5) exactly matched those of the known compound 6-hydroxy-4-methoxycoumarin synthesized in the previous literature [18].It is now isolated for the first time from a natural source.We supplemented its relevant NMR, MS, and UV data here.
The HRESIMS data established the molecular formula of compound 29 as C 11 H 16 O 4 with four degrees of unsaturation.As shown in Table 5, the 1 H and 13 C NMR spectrum displayed signals corresponding to two quaternary carbons (one lactone and one olefinic), five methines (three olefinic and one oxygenated), three methylenes (one oxygenated), and one methyl.The 1 H-1 H COSY correlations from H-4 to H-5 and HMBC correlations from H 2 -4 to C-2 and C-3 as well as H-5 to C-4 established a dihydrofuran-2(3H)-one moiety.The assignment for a spin coupling system of [=CHCH 2 CH 3 ] was identified by 1 H-1 H COSY correlations from H-2 ′′ to H-1 ′′ and H-3 ′′ .Furthermore, a 3,4-dihydroxybut-1-en-1-yl group was assigned by 1 H-1 H COSY correlations from H-2 ′ to H-1 and H-3 ′ , from H-3 ′ to H-4 ′ and 3 ′ -OH, and from H-4 ′ to 4 ′ -OH.The moiety of [=CHCH 2 CH 3 ] spin system was attached to C-3 outside the lactone ring with regard to the HMBC correlations from H-1 ′′ to C-2 and C-4 and from H-2 ′′ to C-3.In addition, the 3,4-dihydroxybut-1-en-1-yl group was bonded to C-5, as proven by the 1 H-1 H COSY correlation from H-1 ′ to H-5 and HMBC correlations from H-1 ′ to C-4 and C-5 and from H-2 ′ to C-5.The E-configuration of double bonds ∆ 1 ′ , 2 ′ was assigned based on the large coupling constants of J = 15.5 Hz, while the cis relation of ∆ 3, 1 ′′ was ascertained by NOESY correlation between H 2 -4 and H-1 ′′ (Figure 8A).
The relative configuration of 29 was determined by NMR calculation with DP4 + analysis.As a result, the experimental NMR data of 29 corresponded to the computed NMR data for (5R*,3'S*)-isomer 29b (99.12% probability, Table S19 in the supplementary material).The absolute configuration of 29 was assigned by ECD calculation.The calculated ECD curves of (5R,3'S)-29 coincided with the measured curve, which demonstrated the absolute configuration of 29 as 5R, 3'S (Figure 8B).Thus, the structure of 29 was established, and it was named talarofuranone A trivially.In addition, based on the comparison of their NMR and optical rotation data with those reported in the literature, the structures of the other sixteen polyketides were identified as phialophoriol (7)   The relative configuration of 29 was determined by NMR calculation with DP4 + analysis.As a result, the experimental NMR data of 29 corresponded to the computed NMR data for (5R*,3 ′ S*)-isomer 29b (99.12% probability, Table S19 in the supplementary material).The absolute configuration of 29 was assigned by ECD calculation.The calculated ECD curves of (5R,3 ′ S)-29 coincided with the measured curve, which demonstrated the absolute configuration of 29 as 5R, 3 ′ S (Figure 8B).Thus, the structure of 29 was established, and it was named talarofuranone A trivially .
Notably, compound 10 displayed significant inhibitory activities against four bacteria, namely E. coli, A. hydrophilia, V. parahaemolyticus, and V. harveyi, with MIC values in
Notably, compound 10 displayed significant inhibitory activities against four bacteria, namely E. coli, A. hydrophilia, V. parahaemolyticus, and V. harveyi, with MIC values in the range of 0.5-1 µg/mL, better than or equivalent to the positive control chloramphenicol (MIC = 0.25-2 µg/mL).Compound 18 showed considerable antibacterial activities against human pathogen E. coli and aquatic pathogenic bacteria V. parahaemolyticus that were comparable to those of the positive control, chloramphenicol.Moreover, compounds 20, 22, and 24 displayed inhibitory effects on the growth of P. aeruginosa with V. vulnificus, A. hydrophilia with V. parahaemolyticus, and A. hydrophilia, respectively, with MIC values like those of chloramphenicol from 0.5 to 4 µg/mL.Unfortunately, all measured compounds did not show any significant antifungal activity at a concentration of 64 µg/mL.
The structure-activity relationship (SAR) for the above polyketides is discussed here.The addition of a ketonic carbonyl or methoxy group in the structures of methylcyclopenta[c] [2]-benzopyran could improve their antibacterial effects (10 vs. 7-9).Then, a comparison of the inhibitory activities of compounds 18-24 indicated that their activities were influenced by the number and position of methyl, hydroxyl, and methoxy groups, as well as the expansion of the lactone ring.Furthermore, a correlation was observed between the acetylation of the side chain and antibacterial efficacy for penicillide-type compounds (30 vs. 31).

General Experimental Procedures
The general experimental procedures in this study were similar to those previously reported [6,14,15,46].

Fungal Material
The fungus strain CS-258 was isolated from the marine mussel sample collected from a cold seep in the northeast of the South China Sea in May 2020, and its strain identification was conducted by a BLAST search in GenBank.This strain sequence data had been uploaded to GenBank to obtain an accession number of No. PP065775.The strain was deposited at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

Fermentation, Extraction, and Isolation
The fresh mycelium of Talaromyces sp.CS-258 was cultured on potato dextrose broth medium for 3 days and then incubated in autoclaved 1 L Erlenmeyer flasks with rice culture medium (70 g rice, 0.2 g corn steep liquor, 0.5 g yeast extract, 0.3 g peptone, 0.6 g gourmet powder, and 100 mL seawater collected from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS) for 30 days at room temperature.After incubation, the EtOAc crude extract was obtained by exhaustive concentration with MeOH and successive extraction with EtOAc/H 2 O four times.
The crude extract was fractionated by silica gel column vacuum liquid chromatography (VLC) elution with increasing polarity gradient of petroleum ether (PE)/EtOAc and CH 2 Cl 2 /MeOH to yield nine fractions (Fr.A-Fr.I).Fr.E was further split by column chromatography (CC) using Sephadex LH-20 to produce three subfractions from E-1 to E-3.Fr.E-1 was rechromatographed over silica gel elution with a slow CH 2 Cl 2 /MeOH gradient from 200:1 to 20:1 to yield three mixtures of E. Fr.F was subjected to reversed-phase CC using Lobar LiChroprep RP-18 from 10% to 90% MeOH-H 2 O to produce three subfractions, Fr.F-1 to F-3.Fr.F-1 was sequentially recrystallized to obtain compound 2 (100.0 mg).Fr.F-2 was subjected to Sephadex LH-20 CC to afford compound 24 (65.9 mg) and two mixed components.Then, the former component was purified by HPLC separation with MeOH-H 2 O (52:48) and identified as 8 (13.7

Antimicrobial Activity Assay
The antimicrobial evaluations against four human pathogens (methicillin-resistant S. aureus, P. aeruginosa, E. coli, and K. pneumonia) and seven aquatic bacteria (V.alginolyticus, A. hydrophilia, M. luteus, V. anguillarum, V. parahaemolyticus, V. vulnificus, and V. harveyi) as well as six plant-pathogenic fungi (C.cornigerum, P. digitatum, P. piricola, V. mali, C. gloeosporioides and F. oxysporum) were carried out in a 96-well microplate as described in our previous reports [6,46,50,51].These microbial strains were provided by the Institute of Oceanology, Chinese Academy of Sciences, by purchase or isolation.Chloramphenicol and amphotericin B served as positive controls against bacteria and fungi, respectively, while DMSO was treated as the negative control.All measurements at various concentrations were performed in triplicate.

Details of Computational Methods
The computational methods for ECD calculation and DP4+ probability analysis were similar to those in the previous papers from our group [6,10,51], with some modifications detailed below.Molecular mechanics using the MM + method was carried out for conformational searches in HyperChem software (Version 8.0, Hypercube, Inc., Gainesville, FL, USA).The energy-minimized conformers were generated and further optimized using DFT calculations at the B3LYP/6-31G(d) level in Gaussian 09 software (Version D.01; Gaussian, Inc.: Wallingford, CT, USA).Frequency calculations were carried out at the same level of theory to confirm the absence of imaginary frequencies and to obtain thermal corrections to the Gibbs free energies.These obtained conformers were subjected to ECD calculations using the TDDFT method at the CAM-B3LYP/TZVP, BH&HLYP/TZVP, or PBE0/TZVP level.The solvent effects of MeOH were evaluated at the same DFT level using the selfconsistent reaction field (SCRF) method with the polarizable continuum model (PCM).The ECD spectrum was generated by the SpecDis program and finally drawn using Origin Pro 8.5 software [52][53][54][55][56][57].The NMR shielding tensors were calculated by the DFT method at the mPW1PW91\6-31+G(d) PCM level in DMSO and then weighted according to Boltzmann's distribution.GIAO (gauge-independent atomic orbital) NMR chemical calculations were performed using an equation described previously.The theoretical shielding tensors ( 1 H and 13 C) and experimental chemical shifts were finally analyzed and compared using DP4+ probability [58][59][60].

The Modified Mosher's Method
The modified Mosher's method was conducted in NMR tubes following the details described in the previous papers [25,61].

Conclusions
In this study, the chemical investigation of cold-seep-derived fungus Talaromyces sp.CS-258 has resulted in the isolation and identification of 32 fungal polyketides including 11 new compounds and 1 new naturally isolated metabolite.Notably, a pair of enantiomers ((±)-11) and two groups of diastereoisomers (13-15 and 25/26) were obtained, the isolation of which presented challenges due to their difficulty in separation from each other.Furthermore, compound 27 was identified as a rare occurring natural iodo-chromone analog.The plausible biogenetic pathways of compounds 1-24, which belong to altenusin or alternariol families with polyketide origin bearing variable tricyclic ring skeletons, were proposed.The results of antimicrobial activities displayed that 11 polyketides exhibited broad-spectrum antibiotic effects against human and aquatic pathogenic bacteria.Among them, compounds 10 and 18 exhibited potent inhibition against E. coli, A. hydrophilia, and V. parahaemolyticus, while 22 and 24 effectively suppressed the growth of A. hydrophilia.These findings highlight the cold-seep-derived fungus Talaromyces sp.CS-258 as a promising source for bioactive metabolites, especially for fungal polyketides, with potential applications as antibiotic agents in medicinal development and agriculture.
Mar. Drugs 2024, 22, x FOR PEER REVIEW 7 of 19hydroxyaltenuene.This was supported by the observation that compounds 13 and 15, with 10aR absolute configurations, displayed positive Cotton effects at 236 nm and negative ones at 282 nm, in contrast to 14 with a 10aS absolute configuration.This was the first isolation of three new epimers of 5-hydroxyaltenuene from the fungus Talaromyces sp.

Compounds 25 and
26 were both obtained as yellow oily substances with similar chromatographic properties in the semipreparative HPLC separation [tR (25) = 14 min, tR (26) = 12 min].The HRESIMS analysis displayed pseudomolecular ion peaks at m/z 255.1228 [M + H] + (calcd for 255.1227) of 25 and at m/z 255.1226 [M + H] + (calcd for 255.1227) of 26, assigning their molecular formula both as C13H18O5 holding five degrees of unsaturation.Their UV spectra exhibited absorption maxima at 209 and 320 nm, revealing structural analogies between 25 and 26.A detailed inspection of 1 H and 13 C NMR spectra of 25

Figure 6 .
Figure 6.Two possible isomers of compound 15 for DP4+ probability analysis.Compounds 25 and 26 were both obtained as yellow oily substances with similar chromatographic properties in the semipreparative HPLC separation [t R (25) = 14 min, t R (26) = 12 min].The HRESIMS analysis displayed pseudomolecular ion peaks at m/z 255.1228 [M + H] + (calcd for 255.1227) of 25 and at m/z 255.1226 [M + H] + (calcd for 255.1227) of 26, assigning their molecular formula both as C 13 H 18 O 5 holding five degrees of unsaturation.Their UV spectra exhibited absorption maxima at 209 and 320 nm, revealing structural analogies between 25 and 26.A detailed inspection of 1 H and 13 C NMR spectra of 25 and 26 (Table4) prominently indicated practically identical signals, suggesting that they shared the same planar structural scaffold.Both 1 H NMR spectral data of 25 and 26 exhibited characteristic resonances ascribed to two olefinic protons (represented for two trisubstituted double bands), three sp 3 -hybridized methines (including two oxygenated), two sp 3 -hybridized methylenes (including one chemically nonequivalent O-substituted), two methyl groups (as a singlet and a doublet), and three exchangeable protons.Analyses of their13 C, DEPT, and HSQC spectrum confirmed the presence of a carbonyl group, two pairs of olefin carbons, two oxygenated methines, one sp 3 -hybridized methine, two methylene groups, two methyls, and one quaternary carbon.The 1 H-1 H COSY and HMBC correlations of 25 and 26 shown in Figure2established the same planar structure, which was similar to nemanecins A-C, three azaphilone analogs isolated from the culture broth of the fungus Nemania sp.BCC 30850[23], indicating that 25 and 26 are a pair of stereoisomers.However, the signal of a singlet methyl in the1 H and 13 C NMR spectra of nemanecins A-C were absent in those of 25 and 26.Instead, signals for a 2-hydroxypropyl group were observed in the NMR spectra of 25 and 26.The above inference was further confirmed by the key 1 H-1 H COSY correlation from H-10 to H-9 and H and 13 C NMR spectra of nemanecins A-C were absent in those of 25 and 26.Instead, signals for a 2-hydroxypropyl group were observed in the NMR spectra of 25 and 26.The above inference was further confirmed by the key 1 H-1 H COSY correlation from H-10 to H-9 and Mar.Drugs 2024, 22, 204 8 of 19

Figure 8 .
Figure 8.The NOESY correlations (A), experimental and calculated ECD spectra (B), and two possible isomers for DP4+ probability analysis (C) of 29.

Figure 8 .
Figure 8.The NOESY correlations (A), experimental and calculated ECD spectra (B), and two possible isomers for DP4+ probability analysis (C) of 29.